The present invention relates generally to cancer treatment. More particularly, the present invention relates to combination therapy using a polypeptide which is an antagonist of the vasoactive intestinal polypeptide (VIP) and a chemotherapeutic agent, preferably in a pharmaceutical composition.
Vasoactive intestinal polypeptide (VIP) is a widely distributed peptide hormone which mediates a variety of physiological responses including gastrointestinal secretion, relaxation of gastrointestinal vascular and respiratory smooth muscle, lipolysis in adipocytes, pituitary hormone secretion, and excitation and hyperthermia after injection into the central nervous system. Vasoactive intestinal peptide is a 28 amino acid peptide with an amidated C-terminus, the peptide results from post transnational processing of a hormone composed of 170 amino acid residues. The VIP peptide has been shown to contain at least two functional regions, a region involved in receptor specific binding and a region involved in biological activity (Gozes and Brenneman, Molecular Neurobiology, 3:201-236 (1989)).
A most striking function of the 28 amino acid peptide, vasoactive intestinal peptide is the ability to promote embryonic growth (see, Gressens, P. et al., Nature, 362:1558-8 (1993)). VIP is secreted by nerve endings, immune cells, and by some neoplastic cells (see, Gozes, I. et al., Current Medicinal Chemistry in press (1999)). Together with its physiological actions, VIP may exert stimulating and trophic effects on neoplastic cells from neuroblastoma (see, Wollman, Y. et al., Brain Res., 624:339-41 (1993)), breast (Zia, H. et al., Cancer Res., 56:3486-9 (1996)) lung (see, Moody, T. W. et al, Proc. Natl., Acad. Sci. USA 90:4345-9 (1993)) and colon cancer (see, Gozes, I. et al., C. Proceedings of the 15th World Congress of Collegium Internatzonale Chirugiae 819 (1996)), inducing its own receptors by feedback mechanisms. In neuroblastoma, the most common solid malignancy in young children, VIP has been shown to have the dual effect of either inducing differentiation or stimulating cell division, depending on the cell line and the time of application. In one human neuroblastoma cell line (I), VIP produced dose-dependent stimulation of mitosis (see, Wollman, Y. et al., Brain Res., 624:339-41 (1993)). In contrast, in the mouse neuroblastoma cell line Neuro2a, VIP inhibited proliferation at concentrations as low as 10xe2x88x9213 M and 10xe2x88x9210 M, respectively. Similarly, in lung cancer, using growth in soft agar as an index of cancer proliferation, VIP induced growth and VIP antagonists inhibited growth small and non-small cell lung cancer (see, Moody, T. W. et al., Proc. Natl, Acad. Sci. USA 90:4345-9 (1993), Moody, T. W. et al., Biomedical Res. 1192, 13 (Suppl. 2) 131).
Gozes, et al. have developed a VIP antagonist that has proven useful for altering the function of the vasoactive intestinal peptide. (See, U.S. Pat. No. 5,217,953 issued to Gozes, et al. (1993)). This VIP antagonist was designed to retain the binding properties of VIP for its receptor, but to lack the amino acid sequence necessary for biological activity. It is believed that biological activity requires, among other factors, a phenylalanine residue at position 6. Amino acids 1-6 of native VIP were therefore replaced by a segment of neurotensin in order to alter the biological activity of native VIP and to change the membrane permeability of the peptide. Three of the six amino acids added in the neurotensin segment are basic. This is in contrast to native VIP which contains no basic residues and only one acidic residue in this region. Indeed, the concept that a tetrapeptide with basic amino acids at both ends and a proline residue adjacent to the N-terminal amino acid is essential for high activity on membrane permeability, has been proven correct for neurotensin and other peptides. As such, the VIP antagonist developed by Gozes, et al. is a hybrid molecule containing an amino acid sequence necessary for VIP receptor binding (i.e., amino acids 7-28 of VIP), and an N-terminal amino acid sequence corresponding to a portion of neurotensin.
Studies have shown that this VIP antagonist effectively antagonizes VIP-associated activity. It has been reported that this VIP antagonist inhibits the growth of VIP receptor bearing tumor cells such as, for example, lung tumor cells (i.e., NSCLC cells). (See, U.S. Pat. No. 5,217,953.)
U.S. Pat. No. 5,565,424, which issued to Gozes, et al. on Oct. 15, 1996, discloses another family of polypeptides which are antagonists of the vasoactive intestinal polypeptide. The VIP antagonists disclosed therein are 10-1000 times more efficacious, i.e., more potent in inhibiting VIP-associated activity than previous VIP antagonists. These superactive VIP antagonists were shown to inhibit cancer growth in lung and glioblastoma cells. Examples of superactive VIP antagonists include amino acid sequences referred to as the xe2x80x9cNL-hybrid VIP antagonistxe2x80x9d, the xe2x80x9cS-NL-hybrid VIP antagonistxe2x80x9d and the xe2x80x9cS-hybrid VIP antagonistxe2x80x9d.
Although the foregoing VIP antagonist and superactive VIP antagonists have been invaluable, there still remains a need in the art for an even more effective cancer treatment. In addition, a treatment is needed which is effective over a broader range of cancers, for solid tumors, and for more advance stages of cancer. The present invention fulfills these and other needs.
The present invention relates to a pharmaceutical composition comprising a vasoactive intestinal polypeptide (VIP) antagonist, a chemotherapeutic agent and a pharmaceutically acceptable carrier. The vasoactive intestinal polypeptide antagonists of the present invention comprise the following amino acid sequence:
R1-Lys-Pro-Arg-Arg-Pro-Tyr-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-X1-Ala-X2-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-AsnNH-R2.
In the above formula, R1 and R2 are independently selected and are functional groups including, but not limited to, the following: hydrogen, C1 to C20 alkyls and C1 to C20 acyls, provided that at least one of R1 or R2 is hydrogen. X1 and X2, in the above formula, are independently selected from the group consisting of naturally occurring amino acids and amino acid analogs or mimetics, provided that X1 is not methionine (SEQ ID NO:1).
Within the scope of the above formula, certain vasoactive intestinal polypeptide antagonists are preferred, namely those in which R1 is H; R2 is H; X1 is a norleucine residue; and X2 is a valine residue (hereinafter referred to as the xe2x80x9cNL-hybrid VIP antagonistxe2x80x9d) (SEQ ID NO:2). Equally preferred are VIP antagonists in which R1 is CH3(CH2)16COxe2x80x94; R2 is H; X1 is a norleucine residue; and X2 is a valine residue (hereinafter referred to as the xe2x80x9cS-NL-hybrid VIP antagonistxe2x80x9d) (SEQ ID NO:3). Also equally preferred are VIP antagonists in which R1 is CH3(CH2)16COxe2x80x94; R2 is H; X1 is a methionine residue; and X2 is a valine residue (hereinafter referred to as the xe2x80x9cS-hybrid VIP antagonistxe2x80x9d) (SEQ ID NO:4). Further equally preferred are VIP antagonists in which R1 is a C1 to C20 alkyl; R2 is H; X1 is a norleucine residue; and X2 is a valine residue (SEQ ID NO:5). In addition, other preferred VIP antagonists are those in which X1 and X2 are amino acids and amino acid analogs or mimetics of hydrophobic character.
It should be noted, however, that R1, R2, X1 and X2 are selected such that the VIP antagonists of the present invention have other than the following amino acid sequence:
Lys-Pro-Arg-Arg-Pro-Tyr-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn (SEQ ID NO:6).
The pharmaceutical compositions of the present invention also comprise a chemotherapeutic agent. In certain aspects, the chemotherapeutic agents of the present invention include, but are not limited to, platinum coordination compounds, topoisomerase inhibitors, antibiotics, antimitotic alkaloids and difluoronucleosides.
In other aspects, the present invention relates to a method of inhibiting the growth of a tumor cell, the method comprising: contacting the tumor cell with an effective amount of the combination of a chemotherapeutic agent and a vasoactive intestinal polypeptide (VIP) antagonist, the VIP antagonist comprising the following amino acid sequence:
R1-Lys-Pro-Arg-Arg-Pro-Tyr-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-X1-Ala-X2-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-AsnNH-R2 
wherein R1, R2, X1 and X2 have been defined above (SEQ ID NOS:1-5), with the proviso that the VIP antagonist does not have the following amino acid sequence:
Lys-Pro-Arg-Arg-Pro-Tyr-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn (SEQ ID NO:6).
In this method, suitable chemotherapeutic agents include, but are not limited to, platinum coordination compounds, topoisomerase inhibitors, antibiotics, antimitotic alkaloids and difluoronucleosides. The method includes the combination of VIP antagonist with the chemotherapeutic agent delivered in a simultaneous manner, in combination therapy wherein the VIP antagonist is administered first, followed by the chemotherapeutic agent, as well as the chemotherapeutic agent being delivered first followed by the VIP antagonist. The present invention includes all such methods of administering and contacting tumor cells.
In further aspects, the present invention relates to a method of inhibiting the growth of a tumor cell in a mammalian subject, the method comprising:
administering to the subject an effective amount of the combination of a chemotherapeutic agent and a vasoactive intestinal polypeptide (VIP) antagonist, the VIP antagonist comprising the following amino acid sequence:
R1-Lys-Pro-Arg-Arg-Pro-Tyr-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-X1-Ala-X2-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-AsnNH-R2 
wherein R1, R2, X1 and X2 have been defined above (SEQ ID NOS:1-5), with the proviso that the VIP antagonist does not have the following amino acid sequence:
Lys-Pro-Arg-Arg-Pro-Tyr-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn (SEQ ID NO:6).
In this aspect, suitable chemotherapeutic agents include, but are not limited to, platinum coordination compounds, topoisomerase inhibitors, antibiotics, antimitotic alkaloids and difluoronucleosides.
In another embodiment, the present invention provides the use for the manufacture of a medicament for the treatment of inhibiting the growth of tumor cells and or cancer therapy. These and other aspects of the present invention will be described in detail hereinbelow.
The term xe2x80x9cindependently selectedxe2x80x9d is used herein to indicate that in a Markush group, for example, R1 and R2 can be identical or different (e.g., R1 and R2 can both be hydrogens, or R1 can be hydrogen and R2 can be C20 alkyl, etc.).
The term xe2x80x9ccontactingxe2x80x9d is used herein interchangeably with the following: combined with, added to, mixed with, passed over, incubated with, flowed over, etc. Moreover, the compositions of present invention can be xe2x80x9cadministeredxe2x80x9d by any conventional methods such as, for example, parenteral, oral, topical and inhalation routes as described herein.
The term xe2x80x9cpharmaceutically acceptable saltxe2x80x9d refers to those salts of compounds which retain the biological effectiveness and properties of the free bases and which are obtained by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. Pharmaceutically acceptable salts include, for example, alkali metal salts, such as sodium and potassium, alkaline earth salts and ammonium salts; The chemotherapeutic agents of the present invention can exist as their pharmaceutically acceptable salts.
xe2x80x9cA combination amount sufficient,xe2x80x9d xe2x80x9can effective combination amountxe2x80x9d xe2x80x9ctherapeutically effective combination amountxe2x80x9d or xe2x80x9can effective amount of the combination ofxe2x80x9d all refer to a combined amount of both the VIP antagonist and the chemotherapeutic agent that is effective to depress, suppress or regress malignant cell growth or that results in amelioration of symptoms associated with cancerous diseases. As used herein, the term xe2x80x9ccombinationxe2x80x9d of VIP antagonist with chemotherapeutic agent means the two compounds can be delivered in a simultaneous manner, in combination therapy wherein the VIP antagonist is administered first, followed by the chemotherapeutic agent, as well as wherein the chemotherapeutic agent is delivered first followed by the VIP antagonist. The desired result can be either a subjective relief of a symptom(s) or an objectively identifiable improvement in the recipient of the dosage, a decrease in tumor size, or a decrease in the rate of growth of cancer cells as noted by the clinician or other qualified observer.
The term xe2x80x9csynergistic effective amountxe2x80x9d refers to a combined amount of both the VIP antagonist and the chemotherapeutic agent that is effective to cause a synergistic effect. Synergy is a chemical phenomenon in which the effectiveness of two active components in a mixture is more than additive, i.e., the effectiveness is greater than the equivalent concentration of either component alone. The effectiveness of the combination therapy of a VIP antagonist and the chemotherapeutic agent is synergistic. Thus, synergism is a result, or function, that is more than the sum of the results, or functions of individual elements.
The terms xe2x80x9ctreating cancer,xe2x80x9d xe2x80x9ctherapy,xe2x80x9d and the like refer generally to any improvement in the mammal having the cancer, wherein the improvement can be ascribed to treatment with the compositions of the present invention. The improvement can be either subjective or objective. For example, if the mammal is human, the patient may note improved vigor or vitality or decreased pain as subjective symptoms of improvement or response to therapy. Alternatively, the clinician may notice a decrease in tumor size or tumor burden based on physical exam, laboratory parameters, tumor markers or radiographic findings. Some laboratory signs that the clinician may observe for response to therapy include normalization of tests, such as white blood cell count, red blood cell count, platelet count, erythrocyte sedimentation rate, and various enzyme levels. Additionally, the clinician may observe a decrease in a detectable tumor marker. Alternatively, other tests can be used to evaluate objective improvement, such as sonograms, nuclear magnetic resonance testing and positron emissions testing.
xe2x80x9cInhibiting the growth of tumor cellsxe2x80x9d can be evaluated by any accepted method of measuring whether growth of the tumor cells has been slowed or diminished. This includes direct observation and indirect evaluation, such as subjective symptoms or objective signs as discussed above.
The term xe2x80x9camino acidxe2x80x9d refers to naturally occurring and synthetic amino acids as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acid. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, xcex1-carboxyglutamate and O-phosphoserine. Amino acids analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid i.e., an xcex1-carbon that is bound to a hydrogen, a carboxyl group, an amino group and an R-group, e.g. homoserine, norleucine, methionine sulfoxide and methionine methyl sulfonium. Such analogs have modified R groups (e.g. norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid memetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid but that functions in a manner similar to an naturally occurring amino acid.
Some of the amino acids referred to herein are described by shorthand designations as follows: